Acta Biologica Hungarica 68(4), pp. 443–452 (2017) DOI: 10.1556/018.68.2017.4.9
IN VIVO SCREENING OF DITERPENE ALKALOIDS USING BDELLOID ROTIFER ASSAYS
Tivadar Kiss,1,2 LiLLa Mácsai,3 Dezső Csupor,1,2 * and zsolt lászló Datki3,*
1University of Szeged, Faculty of Pharmacy, Department of Pharmacognosy, Eötvös u. 6, H-6720 Szeged, Hungary
2University of Szeged, Interdisciplinary Centre for Natural Products, Eötvös u. 6, H-6720 Szeged, Hungary
3University of Szeged, Faculty of Medicine, Department of Psychiatry, Kálvária sgt. 57, H-6725 Szeged, Hungary
(Received: May 17, 2017; accepted: June 13, 2017)
The group of diterpene alkaloids contains numerous compounds with complex chemistry and diverse pharmacological activities. Beside toxicity, these compounds possess activity on the cardiovascular sys- tem, tumor cell lines and nervous system. The pharmacological properties have been described using in vitro and in vivo techniques; however, the bioactivities of many compounds have not thoroughly been studied. Here we report on the in vivo evaluation of ten diterpene alkaloids using bdelloid rotifer assays.
Napelline exerted toxic effects on rotifers, while wide tolerance range was observed for other investi- gated compounds. Weak toxicity of songorine is supported by our experiment. Toxicological data for senbusine A, senbusine C, septentrioidine and hetisinone are reported for the first time.
Keywords: Diterpenoid alkaloids – toxicity – Aconitum – rotifers – Philodina acuticornis odiosa
INTRODUCTION
Diterpene alkaloids are secondary plant metabolites with a wide range of biological activity. The structures and substitution patterns of these compounds are highly diverse. Diterpene alkaloids are classified in order to the basic skeleton’s carbon atom number (C18 – bisnorditerpene alkaloids, C19 – norditerpene alkaloids, C20 – diterpene alkaloids) and by their ester groups.
Most of these compounds are highly toxic, while some of them exert beneficial pharmacological effects. Aconitine (3) is a well-known cardiotoxic compound. In various in vivo experiments, it is used for inducing arrhythmia. In vitro studies pro- vide explanation for this effect: aconitine (3), together with other arrhythmogenic alkaloids (hypaconitine, mesaconitine) activates voltage-dependent Na+ channels at their resting potential and inhibit their inactivation, resulting in a final inexcitability of the cells [3]. Interestingly, among diterpene alkaloids there are compounds with opposite, antiarrhythmic activity. Napelline (8) showed to exert low antiarrhythmic activity in rats [31], while delavaconitine (2) inhibited aconitine-induced arrhythmia
* Corresponding author; e-mail address: csupor.dezso@pharmacognosy.hu; datkizsolt@gmail.com
THE DOI OF THE PUBLISHED VERSION: 10.1556/018.68.2017.4.9
[21]. In vitro study revealed the activity of acontine (3) and hetisione (9) on Nav1.2 sodium channel. Certain alkaloids act as competitive antagonists of aconitine (3). In the mechanism of the cardiac activity hERG and GIRK potassium channels are also involved. Aconitine (3) exerts cardiotoxicity through high hERG channel inhibition, while there is no such safety issue in case of songorine (10), because of its low inhibitory potential. Similarly, aconosine (1), neoline (4), senbusine A (5), senbusine C (6) and napelline (8) are having low potential on hERG channels. A comprehensive investigation of these compounds led to the development of a new group of antiar- rhythmic drugs (e.g. lappaconitine hydrobromide) [28].
Among diterpene alkaloids there are many toxic compounds, however the toxicity does not exclude possible pharmacophore nature [4]. Furthermore, the pharmaco- logical activities of several diterpene alkaloids are either poorly or not investigated at all. The aim of the current study was to investigate toxicity and pharmacological activity of bisnorditerpene alkaloids: aconosine (1), delavaconitine (2); norditerpene alkaloids: aconitine (3), neoline (4), senbusine A (5), senbusine C (6), septentrioidine (7); diterpene alkaloids: napelline (8), hetisinone (9) and songorine (10); using high- throughput viability and non-invasive bdelloid rotifer assay.
The pharmacological literature of these compounds revealed several different bio- activities beyond cardiac effects. The arrhythmia-inducing aconitine (3) showed to be potent apoptotic agent against pancreatic cancer (MIA PaCa-2, PANC-1) [15], breast cancer (MDA-MB-231) [12] and melanoma (B16 cell line) [9]. Neoline (4) and sep- tentriodine (7) have been exerting cytotoxic activity in vitro [14, 17], however their effect is not supported by in vivo experiments.
Napelline (8) and songorine (10) exerted interesting effects in vitro and in vivo, as well. These alkaloids activate mesenchymal stem cells thereby increasing wound healing [33, 34]. The anti-inflammatory effect of both compounds was comparable to diclofenac, in histamine-induced acute inflammation model in mice [20]. In rats, intraperitoneal administration of both compounds was shown to reduce painful reac- tion induced by acetic acid. However, the mechanism of analgesia is different. In case of songorine (10) it is assumed that the opioid system is involved [19]. Hetisinone (9) exerts anticholinesterase activity in vitro [1]. Several studies have reported central nervous system activities of these two compounds. Both exerted antidepressant activ- ity in various animal models and showed to be effective stress modulators [18]. An extensive literature review, based on numerous in vivo and in vitro experimental data about diterpene alkaloids in general, lead to the understanding of the structure-activ- ity relationship in case of cardiac [11], anticancer [13, 40] and analgesic [30] activity.
The conclusion is contradictory in case of central nervous system activity. For exam- ple, songorine (10) acts in vivo as agonist on D2 [2] and GABAA receptors [16], however in vitro it exerts antagonistic activity [33]. In case of other alkaloids involved in the present experiments, no pharmacological data are available.
The in vivo studies, using living organisms are suitable to generat the most accurate data for pharmacological activities and toxicity assessment. Acceptance by the scien- tific community, reproducibility, sensitivity, economic are criteria which in vivo stud- ies have to meet. Rotifers are widely used in toxicity assessment and they fulfill all
aforementioned requirements [25]. Various Rotifera species have been used for method development, however, none of these were applied for the examination of diterpene alkaloids [25, 27].
MATERIALS AND METHODS Diterpene-alkaloids
The examined diterpene alkaloids (Fig. 1) were isolated previously from A. toxicum Rchb. (1–2), A. napellus L. subsp. firmum (3–6, 8), A. vulparia Rchb. (7) and A. anthora L. (9–10) [7, 8]. The purity (≥95%) of the isolated compounds was con- firmed by HPLC and 1H NMR spectroscopy.
Fig. 1. Chemical structures of examined compounds
Animals
Philodina acuticornis odiosa (PA) and Cladophora aegagropila (CA) were obtained from a Hungarian aquaristic. Rotifer culturing methods were developed based on previous literature [23]. The animals were cultured in standard medium (SM), in data a supervised and semi-sterile environment. Clear cultures of PA were kept in stand- ardized cell culturing flasks (cat. no.: 83.3910.302, Sarstedt AG & Co., Germany), at 25 °C and under a light/dark cycle of 12:12 hours. The exact composition of SM, matrix and food, along with the pattern of feeding and culturing were described at length by Oláh et al. [23].
Rotifers approximately 5 day after hatching (determined by body size; length 220 ± 10 µm and width 60 ± 5 µm), 1–2 days before the beginning of the reproductive stage, were chosen. The selection process followed the harvesting protocols, published in ref [23].
Non-invasive experimental monitoring assays
The methodical protocols have been reported in our previous paper [23], therefore in this manuscript we only intend to give an overview of the applied techniques.
After 24 h of the standard isolation process, the rotifers were treated in a 384 well- plate (cat. no.: 3657, Corning Inc., USA; n = 32) with diterpene alkaloids (1–8). For this in vivo experiment stock solutions were prepared with 1% aqueous DMSO. The stock solutions were added to standard media reaching 100 μM final concentrations for diterpene alkaloid and 0.1% DMSO content. Untreated control group (UC) were grown in SM, while control group (C) were kept in SM containing 0.1% DMSO. The effect exerted by diterpene alkaloids were compared to C. This treatment period lasted for 72 hours (toxicity interval), without feeding. From the fourth day began the daily monitoring period under restricted calorie state (yeast solution, 100 µg/mL).
The viability of rotifers was assessed with three different assays utilizing video recordings with a Nikon D5500 DSLR camera.
Toxicity and survival lifespan (TSL) assay
The impact of the test compounds on the lifespan of unfed PA rotifers was assessed.
The morphological viability markers, chosen for evaluation were adapted from Poeggeler et al. [24] were extended and defined in our previous work [23].
Body size index (BSI) measurement
Rotifers never stop growing; therefore the length/width of the animal in correlation with time (body size index; BSI) increases continuously within the species-specific limit.
Fig. 2. TSL: toxicity and survival lifespan (A), BSI: body size index (B), MCF: mastax contraction frequency (C), CRC: cellular reduction capacity (D). Values are the mean ± SEM; *p < 0.05, **p < 0.01; ***p < 0.001
Mastax contraction frequency (MCF) assay
The mastax (pharynx) is part of the digestive system. To evaluate and standardize the viability of one-housed rotifers in our experiments, we developed the MCF (contrac- tion/sec) as a quantitative viability marker.
Invasive experimental monitoring assay Cellular reduction capacity (CRC) assay
The CRC gives information about the scale of reduction capacity and oxidative stress triggered by treatment in rotifers. For measuring the EZ4U Cell Proliferation Assay (non-radioactive cell proliferation, cytotoxicity and reduction capacity assay with XTT solution) was used (cat. no.: BI-5000; Biomedica Hungary). The absorbance was measured by a microplate-reader (Spectramax 384, Molecular Science, Hungary) set at 491 nm with 630 nm as a background reference.
Data analyses
Data are presented as means ± SEM. Statistical evaluation and curve fitting were per- formed with Prism 7.0 (GraphPad Software) and with SPSS 23.0 software for Windows, using One-way ANOVA with post hoc Bonferroni test.
RESULTS
Ten diterpene alkaloids, belonging to the bisnor- (1, 2), nor- (3–8) and diterpene (9, 10) were evaluated for their effects on PA viability (Table 1).
All compounds, except napelline (8), increased the toxic survival lifespan value (TSL). Senbusine A (5) and hetisinone (9) resulted in significantly higher TSL assay values (Fig. 2A). Most of the compounds (1, 3–6, 8–10) increased significantly the body size index (BSI) compared to the control group (Fig. 2B). Napelline (8) had slightly decreasing effect on BSI value. The mastax contraction frequency (MFC) was significantly decreased by napelline (8), and increased by aconosine (1) and son- gorine (10) (Fig. 2C). Significantly elevated cellular reduction capacity (CRC) was observed in case of treatment with aconosine (1), aconitine (3), senbusine C (6) and songorine (10), while napelline (8) significantly lowered this value (Fig 2D).
DISCUSSION
We have measured the universal aspects of rotifers phenotype, to characterize Philodina acuticornis odiosa in terms of survival, health and behaviour. Decrease or increase in the experimental parameters of the rotifers are in correlation with the physiological state of individuals.
The investigated compounds proved to be non-toxic in these assays, except napel- line (8), which reduced viability parameters. The wide tolerance range of alkaloids – including C18, C19 and C20 diterpene alkaloids – are reported for the first time. This conclusion is supported by the results of toxicity and survival lifespan (TSL) which provides mortality rate and the body size index (BSI). One norditerpenoid (5) and one diterpenoid (9) even increased the TSL. Only napelline (8) showed to be toxic by significantly decreasing the lifespan. Most of the investigated alkaloids increased the BSI, except napelline (8) which slightly decreased this value. The elevated mastax contraction frequency values showed no toxicity symptoms, since aconosine (1) and songorine (10) upregulated, while napelline (8) downregulated the metabolic state of the examined animals. The cellular reduction capacity gives information about the degree of reduction capacity and oxidative stress triggered by treatment. All com- pounds elevated cellular reduction capacity, except napelline (8).
Most of the examined compounds have been poorly investigated for their toxicity (Table 2). The median lethal dose (LD50) in mice were available only for compound 1–4, 8 and 10. According to these data, the most toxic compound is aconitine (3), while songorine (10) is the least toxic. The weak toxicity of songorine (10) is also supported by our experiment. The well-known aconitine (3) possesses high toxicity
Table 1
Viability parameters (mean ± SEM, n = 32)
Compound TSL BSI MCF CRC
Aconosine (1) 107.1±5.14 136.9±2.92 115.5±0.91 140.1±6.09
Delavaconitine (2) 109.8±6.64 113.9±3.19 102.6±1.68 115.9±6.57
Aconitine (3) 108.5±5.18 116.5±3.00 101.7±1.78 119.9±4.96
Neoline (4) 109.3±7.04 117.4±3.45 102.0±2.05 116.7±3.96
Senbusine A (5) 110.9±6.41 112.2±2.73 98.9±2.18 115.5±5.54
Senbusine C (6) 107.1±5.42 123.1±2.43 101.4±2.03 129.1±5.74
Septentriodine (7) 109.6±6.12 114.5±3.05 102.9±1.79 111.1±4.79
Napelline (8) 97.6±5.24 98.7±2.76 93.9±1.87 92.0±2.78
Hetisinone (9) 110.7±5.90 113.9±3.00 103.9 ±1.78 116.4±6.35
Songorine (10) 108.4±6.28 159.2±3.49 114.6±0.74 151.8±3.61
TSL – toxicity and survival lifespan; BSI – body size index; MCF – mastax contraction frequency; CRC – cel- lular reduction capacity.
in vivo in rodents and mammalians, as well [26]. However, in our assay, napelline (8) showed to be the most toxic.
Since there is yet no toxicity data published for compounds 5–7 and 9, our present can be considered a priori report in vivo toxicity results.
In summary, this in vivo screening system, with four different methods, made pos- sible to measure various conditions at different sensitivity levels of detection inde- pendently and/or simultaneously, providing a reliable and highly replicable screening method in pharmaceutical research.
ACKNOWLEDGEMENTS
Financial support from the National Research, Development and Innovation Office (OTKA K115796), Economic Development and Innovation Operative Programmw GINOP-2.3.2-15-2016-00012 and János Bolyai Research Scholarship of the Hungarian Academy of Sciences are gratefully acknowledged.
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